Carrier for targeting nerve cells

ABSTRACT

The present invention relates to a transport protein which can be obtained by modifying the heavy chain of the neurotoxin formed by  Clostridium botulinum  wherein (i) the protein binds specifically to nerve cells with a higher or lower affinity as the native neurotoxin; (ii) the protein has an increased or reduced neurotoxicity compared to the native neurotoxin, the neurotoxicity being preferably determined in the hemidiaphragm assay; and/or (iii) the protein comprises a lower affinity against neutralizing antibodies compared to the native neurotoxin. The invention also relates to methods for producing the same and the use thereof in cosmetic and pharmaceutical compositions.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/867,740, filed Apr. 22, 2013; which is a continuation of U.S. patentapplication Ser. No. 11/919,302, having a 371 filing date of Feb. 29,2008; which is a 371 application of PCT/EP2006/003896, filed on Apr. 26,2006; which claims priority from German Patent Application No.102005019302.1, filed on Apr. 26, 2005, each of which is incorporated byreference in its entirety herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 13, 2016, isnamed 120574-00203_Substitute_Seq_List.txt and is 292,637 bytes in size.

The present invention relates to a transport protein which binds toneurons with a higher or lower affinity than the neurotoxin formed byClostridium botulinum. The transport protein is preferably absorbed byreceptor-mediated endocytosis. This protein is used as a transportingmeans translocating other chemical substances (e.g. proteases) from theacid endosomal compartment into the cytosol of neurons which are unablephysiologically to penetrate into the cytosol of nerve cells through theplasma membrane. The present invention relates, in particular, to theuse of a transport protein for the introduction of inhibitors of therelease of neurotransmitters.

Nerve cells release transmitter substances by exocytosis. The fusion ofthe membranes of intracellular vesicles with the plasma membrane isreferred to as exocytosis. In the course of this process the vesicularcontents is simultaneously released into the synaptic gap. The fusion ofthe two membranes is regulated by calcium, reacting with the proteinsynaptotagmin. Jointly with other co-factors synaptotagmin controls thestatus of three so-called fusion proteins, SNAP-25, synaptobrevin 2 andsyntaxin 1A. While syntaxin 1A and synaptobrevin 2 are integrated intothe plasma and/or vesicle membrane, SNAP-25 binds only lightly to theplasma membrane. To the extent that the intracellular calciumconcentration increases, the three proteins bind to one another, bothmembranes approaching one another and subsequently fusing together. Inthe case of cholinergic neurons acetyl choline is released, causingmuscle contractions, perspiration and other cholinergically provokedreactions.

The above mentioned fusion proteins are the target molecules(substrates) of the light chain (LC) of the clostridial neurotoxins,formed by the bacteria C. botulinum, C. butyricum, C. baratii and C.tetani.

The anaerobic, gram-positive bacterium C. botulinum produces sevendifferent serotypes of the clostridial neurotoxins. The latter arereferred to as the Botulinus neurotoxins (BoNT/A to BoNT/G). Amongthese, in particular BoNT/A and BoNT/B cause a neuroparalytic disorderin humans and animals, referred to as botulism. The spores of C.botulinum can be found in the soil, but may also develop in incorrectlysterilised and sealed home-made food preserves, to which many cases ofbotulism are attributed.

BoNT/A is the most active of all known biological substances. As littleas 5-6 pg of purified BoNT/A represents an MLD (Multiple Lethal Dose).One unit (Engl.: Unit, U) of BoNT/A is defined as the MLD, killing halfof the female Swiss Webster mice, each weighing 18-20 g, afterintraperitoneal injection. Seven immunologically different BoNTs werecharacterised. They are denoted as BoNT/A, B, C1, D, E, F and G and maybe distinguished by neutralisation with serotype-specific antibodies.The different serotypes of BoNTs differ in affected animal species withregard to severity and duration of the paralysis caused. Thus, withregard to paralysis, BoNT/A is 500 times more potent in rats forexample, than BoNT/B. In addition, BoNT/B has proved to be non-toxic inprimates at a dosage of 480 U/kg of body weight. The same quantity ofBoNT/A corresponds to 12 times the lethal dose of this substance inprimates. On the other hand, the duration of paralysis after BoNT/Ainjection in mice is ten times longer than after injection of BoNT/E.

BoNTs are used for treating neuromuscular disorders, characterised byhyperactivity in skeleton muscles, caused by pathologically overactiveperipheral nerves. BoNT/A has been approved by the U.S. Food and DrugAdministration for treating blepharospasm, strabism, hyperhidrosis,wrinkles and hemi-facial spasms. Compared to BoNT/A the remaining BoNTserotypes are evidently less efficacious and manifest a shorter durationof efficacy. Clinical effects of BoNT/A administeredperipheral-intramuscularly are usually noticeable within a week.

The duration of symptom suppression by one single intramuscularinjection of BoNT/A is normally about three to six months.

The clostridial neurotoxins specifically hydrolyse different proteins ofthe fusion apparatus. BoNT/A, C1 and E break up SNAP-25, while BoNT/B,D, F, G as well as tetanus neurotoxin (TeNT) attack thevesicle-associated membrane protein (VAMP) 2—also referred to assynaptobrevin 2—. BoNT/C1 furthermore breaks up syntaxin 1A.

The Clostridium bacteria release the neurotoxins as single-chainpolypeptides each having 1251 to 1315 amino acids. Thereafter endogenousproteases split each of these proteins at a defined location into 2chains each (‘nicking’), the two chains however remaining interlinked bya disulphide-bridge. These dual-chain proteins are referred to asholotoxins (see Shone et al. (1985), Eur. J. Biochem. 151, 75-82). Thetwo chains have different functions. While the smaller fragment, thelight chain (light chain=LC), represents a Zn²⁺-dependent endoprotease,the larger unit (heavy chain=HC) represents the transporting means ofthe light chain. By treating the HC with endopeptidases two 50 kDafragments were brought about (see Gimenez et al. (1993), J. ProteinChem. 12, 351-363). The amino-terminal half (H_(N)-fragment) integratesinto membranes at a low pH-value and translocates the LC into thecytosol of the nerve cell. The carboxyl-terminal half (H_(c)-fragment)binds to complex polysialogangliosides, occurring exclusively in nervecell membranes and to protein receptors identified only partially todate (Halpern et al. (1993), Curr Top Microbial Immunol 195, 221-241).The latter explains the high neuroselectivity of the clostridialneurotoxins. Crystalline structures confirm that BoNT/A disposes ofthree domains, which may be harmonised by the three steps of the actionmechanism (see Lacy et al. (1998), Nat. Struct. Biol. 5, 898-902).Moreover, these data give rise to the conclusion that within theH_(C)-fragment two autonomous subunits (sub-domains) exist of 25 kDaeach. The first proof for the existence of the two functionalsub-domains was brought about by the amino-terminal (H_(CN)) and thecarboxyl-terminal half (H_(CC)) of the H_(C)-fragment of the TeNT, whichwere expressed in recombinant form and which revealed that the H_(CC)-,but not the H_(CN) domain binds to neurons (see Herreros et al. (2000),Biochem. J. 347, 199-204). At a later stage, a single gangliosidebinding site within the H_(CC)-domains of BoNT/A and B was localised andcharacterised (see Rummel et al. (2004), Mol. Microbiol. 51, 631-643).The site for binding the synaptotagmin I and II, identified as proteinreceptor for BoNT/B and G, could likewise be restricted to the region ofthe H_(CC)-domains of BoNT/B and G (see Rummel et al. (2004), J BiolChem 279, 30865-70). The document does, however, not disclose the aminoacids relevant to the binding pocket of BoNT/B and G.

Under physiological conditions the HC with the H_(C)-fragment binds toneuronal gangliosides, is absorbed inside the cell by receptor-mediatedendocytosis and reaches the natural vesicle circulation via theendosomal compartment. In the acid medium of the early endosomes, theH_(N) fragment penetrates into the vesicle membrane and forms a pore.Each substance (X), linked to the HC via a disulphide bridge, will besplit off the HC by intracellular redox systems, gaining access to thedisulphide bridge and reducing it. X will ultimately appear in thecytosol.

In the case of the clostridial neurotoxins the HC is the carrier of anLC, splitting its specific substrate in the cytosol in the final step.The cycle of complex formation and dissociation of the fusion proteinsis interrupted and the release of acetyl choline is consequentlyinhibited. As a result thereof, striated muscles are paralysed and sweatglands cease their secretion. The active period of the individual BoNTserotypes differs and depends on the presence of intact LC in thecytosol. As all neurons possess receptors for clostridial neurotoxins,it is not only the release of acetyl choline which may be affected, butpotentially also the release of the substance P, of noradrenalin, GABA,glycine, endorphin and other transmitters and hormones.

That the cholinergic transmission is blocked preferentially, may beexplained by the fact that the HC in the periphery enters into theneuron. Central synapses are protected by the blood-brain-barrier, whichcannot be surmounted by proteins.

In a ligand-receptor study specific amino acid residues were substitutedwithin the H_(CC)-domain of BoNT/B and G in order to identify andcharacterise the binding pocket of the protein receptor in order to thusmodify the affinity to the protein receptor. The affinity of the mutatedH_(C)-fragments of BoNT/B and G were determined by synaptotagmin ingluthathione-S-transferase-(GST)-pull-down experiments. The HCexhibiting the same mutations was subsequently coupled to LC-B or,respectively LC-G. The potency of these constructs was analysed by meansof the isolated nerve-muscle-preparation of the mouse(Hemi-Diaphragm-Assay=HDA). In this preparation the Nervus phrenicus isto be found, which consists of cholinergic motor neurons and representsthe most important physiological object of clostridial neurotoxins.Subsequently, individual amino acids were substituted in theH_(CC)-domain of BoNT/A in a depression, located analogously to the siteof the protein-receptor-binding pockets in BoNT/B and G. The full-lengthBoNT/A single mutants were subsequently likewise analysed by HDA withregard to modified potency, giving indications as to modifiedligand-protein-receptor-interactions.

In the more recent past, the BoNT/A complex, also referred to asprogenitor toxin A, has been used for treating motor dystonias, as wellas for attenuating excessive sympathetic activity (see Benecke et al.(1995), Akt. Neurol. 22, 209ff) and for alleviating pain and migraine(see Sycha et al. (2004), J. Neurol. 251, 19-30). This complex consistsof the neurotoxin, various haemagglutinines and a non-toxic,non-haemagglutinating protein. The complex dissociates within a fewminutes at physiological pH. The resultant neurotoxin is the soleingredient of the complex which is therapeutically relevant and bringsabout an alleviation of the symptoms. Since the underlying neurologicalillness is not cured, the complex needs to be injected again atintervals of three to four months. Depending on the quantity of theinjected foreign protein, some patients develop specificBoNT/A-antibodies. These patients become resistant to the neurotoxin.Once antigen-sensitive cells have recognised the neurotoxin andantibodies have been formed, the relevant memory cells are conservedover years. For this reason it is important to treat the patients withpreparations of the highest possible activity at the lowest possibledosage. The preparations should furthermore not contain any furtherproteins of bacterial origin, since these may act as immuno-adjuvants.Such substances attract macrophages, which recognise both theimmuno-adjuvants as well as the neurotoxins, presenting them to thelymphocytes, which thereupon respond by forming immunoglobulins.Consequently, only products of extreme purity, not containing anyforeign proteins, should be used for therapy. The resistance of patientsto the neurotoxin, viewed at a molecular level, is based predominantlyon the presence of neutralising antibodies.

In what follows, the present invention proposes a transport protein(Trapo), which is able to overcome the above described problems of themethods known to date.

This object was obtained by a new transport protein, which can beobtained by modifying the heavy chain of the neurotoxin formed byClostridium botulinum, wherein

-   -   (i) the protein binds to nerve cells with a higher or lower        affinity than the native neurotoxin;    -   (ii) the protein has an increased or reduced neurotoxicity        compared to the native neurotoxin, the neurotoxicity being        preferably determined in the hemidiaphragm assay; and/or,    -   (iii) compared to the native neurotoxin, the protein exhibits a        lower affinity in relation to neutralising antibodies.

According to a preferred embodiment of the present invention, atransport protein is provided which binds to nerve cells with a higheror lower affinity than the native neurotoxin formed by C. botulinum.

According to a further preferred embodiment of the present invention, atransport protein is provided which is obtained by modifying the HC ofthe neurotoxin formed by C. botulinum, the protein binding specificallyto nerve cells with a higher or lower affinity than the nativeneurotoxin. The transport protein is preferably absorbed by these cellsby endocytosis.

In addition, according to a further preferred embodiment, a transportprotein is provided which is obtained by modifying the HC of theneurotoxin formed by C. botulinum, the protein, by substitutingsurface-exposed amino acids, in particular on the ganglioside- andprotein-receptor binding pockets of the binding of neutralisingantibodies no longer being accessible.

In what follows, terms are defined, which are to be understood in thecontext of the present application.

“Binding to nerve cells with a higher or lower affinity than nativeneurotoxin”. The native neurotoxin is in this case the native neurotoxinof C. botulinum. Preferably, the native neurotoxin is in this contextBotulinus neurotoxin A and/or Botulinus neurotoxin B and/or Botulinusneurotoxin G from C. botulinum. The Botulinus neurotoxin prepared inrecombinant form from E. coli, which, inter alia, contains the aminoacid sequence identical to the native Botulinus neurotoxin, acts in apharmacologically identical manner to the native Botulinus neurotoxinand is referred to as recombinant Botulinus neurotoxin wild type. Thenerve cells mentioned in this case are cholinergic motor neurons.Preferably, the transport protein binds specifically to the moleculesassociated with the plasma membrane, transmembrane proteins, synapticvesicle proteins, a protein of the synaptotagmin family or the synapticvesicle glycoproteins 2 (SV2), preferably synaptotagmin I and/orsynaptotagmin II and/or SV2A, SV2B or SV2C, particularly preferablyhuman synaptotagmin I and/or human synaptotagmin II and/or human SV2A,SV2B or SV2C. Bonding is preferably determined in vitro. Particularlypreferably, the determination is performed by using aGST-pull-down-assay, elucidated in detail in the examples.

“the protein has an increased or reduced neurotoxicity compared to thenative neurotoxin”. The native neurotoxin is in this case the nativeneurotoxin of C. botulinum. Preferably, the native neurotoxin is in thiscontext the Botulinus neurotoxin A and/or Botulinus neurotoxin B and/orBotulinus neurotoxin G from C. botulinum. The Botulinus neurotoxinprepared in recombinant form from E. coli, which, inter alia, containsthe amino acid sequence identical to the native Botulinus neurotoxin,acts in a pharmacologically identical manner to the native Botulinusneurotoxin and is referred to as recombinant Botulinus neurotoxin wildtype. The nerve cells mentioned in this case are cholinergic motorneurons. The neurotoxicity is preferably determined with the aid of theHemi-Diaphragm-Assay (HDA) known in the art. The neurotoxicity of themutants can preferably be determined as described by Habermann et at,Naunyn Schmiedeberg's Arch. Pharmacol. 311 (1980), 33-40.

“Neutralising antibodies”. Neutralising antibodies directed againstBotulinus neurotoxin are known (Göschel H, Wohlfarth K, Frevert J,Dengler R, Bigalke H. Botulinum A toxin therapy: neutralizing andnonneutralizing antibodies-therapeutic consequences, Exp. Neurol. 1997September; 147(1):96-102). It was found that antibodies neutralisingneurotoxin interact, in particular, with the active centres such as, forexample, the ganglioside- and protein-receptor binding pockets withinthe H_(CC)-domain of the neurotoxin. If the surfaces surrounding thebinding pockets are modified in the neurotoxin by amino acidsubstitutions without negatively impairing their functionality, theneutralising antibodies lose their binding sites and the mutatedneurotoxin is no longer neutralised.

The term “modification of the heavy chain of the neurotoxin formed by C.botulinum.” The amino acid and/or nucleic acid sequence of the heavychain (HC) of the neurotoxin formed by C. botulinum are generallyavailable from publicly accessible databases, for each of the knownserotypes A to G (also refer to table 1). Modification includes in thiscontext that at least one amino acid is deleted, added, inserted intothe amino acid sequence, or that at least one amino acid of the nativeneurotoxin is substituted by another naturally occurring or notnaturally occurring amino acid and/or that one amino acid in the givenamino acid sequence is modified post-translationally. Post-translationalmodifications include in this context glycosylations, acetylations,acylations, de-aminations, phosphorylisations, isoprenylisations,glycosyl phosphatidyl inositolisations and further modifications knownto the person skilled in the art.

The HC of the neurotoxin formed by C. botulinum includes threesub-domains, i.e. the amino-terminal 50 kDa-sized translocation domainH_(N), the 25 kDa H_(CN)-domain following thereon, and thecarboxyl-terminally situated 25 kDa H_(CC)-domain. Together, the H_(CN)-and H_(CC)-domains are denoted as H_(C)-fragment. The correspondingamino acid sections of the respective sub-domains for the individualserotypes and their variations are apparent from Table 1.

“Ganglioside Receptor”

The HCs of the Botulinus neurotoxins exhibit a high affinity toperipheral nerve cells which is mediated predominantly by theinteraction with complex polysialogangliosides—these are glycolipidsconsisting of more than one sialic acid—(Halpem et al. (1995), Curr.Top. Microbiol. Immunol. 195, 221-41; WO 2006/02707). The LCs bound tothem consequently only reach this cell type and become active in thesecells only. BoNT/A and B merely bind one ganglioside GT1b molecule.

In the case of BoNT/B and BoNT/G the protein receptors are synaptotagminI and synaptotagmin II. In the case of BoNT/A the protein receptors arethe synaptic vesicles glycoproteins 2 (SV2), preferably SV2A, SV2B andSV2C.

At present 13 isoforms pertaining to the family of synaptotagmins areknown. All are characterised by two carboxyl-terminal Ca²⁺ bindingC2-domains, a central transmembrane domain (TMD), which anchors thesynaptotagmin in the synaptic vesicle membrane, and an amino terminushaving different lengths. After the Ca²⁺ inflow the fusion of thesynaptic vesicle with the plasma membrane is initiated, whereupon theintraluminal amino terminus of the synaptotagmin is presentedextracellularly and is available as receptor anchor for BoNT/B and G.Analogously thereto, the fourth luminal domain of the SV2 isoforms isavailable extracellularly, after exocytosis, for the interaction withBoNT/A.

The character of individual amino acids of the binding pocket was somodified by specific mutagenesis that binding to a protein receptor isrendered more difficult or is inhibited. For this purpose, theH_(C)-fragments of BoNT/B and BoNT/G were expressed in E. coli andisolated in the postulated binding pocket in recombinant form as wildtype or with individual amino acid substitutions(mutations/substitutions). For a GST-pull-down-assay, in order to studythe interaction in vitro between BoNT/B and BoNT/G as well as betweensynaptotagmin I and synaptotagmin II, the respectiveGST-synaptotagmin-fusion protein was incubated with different quantitiesof the respective H_(C)-fragment of BoNT/B or, respectively, BoNT/G anda phase separation was performed. Free H_(C)-fragment remained in theseparated supematant while bound BoNT H_(C)-fragment could be detectedin the solid phase, together with GST-synaptotagmin-fusion protein.Substitution of the respective H_(C)-fragments by the full-length BoNT/Band G in the GST-pull-down assay showed the same results.

It was found in this context that the BoNT/B wild type only binds in thepresence of complex gangliosides and synaptotagmin I with transmembranedomain, while synaptotagmin II binds both with or without transmembranedomain as well as in the presence or absence of complex gangliosides. Byspecifically substituting amino acids within the protein receptorbinding site of BoNT/B it was possible to significantly increase ordecrease the interaction between both synaptotagmin molecules (FIG. 1).

Furthermore, it was shown for the BoNT/G wild type that binding tosynaptotagmin I and synaptotagmin II, in each case with or withouttransmembrane domain, is taking place both in the presence as well as inthe absence of complex gangliosides. By specifically substituting aminoacids homologous to BoNT/B, within the protein receptor binding site ofBoNT/G, it was possible to significantly increase or decrease theinteraction between both synaptotagmin molecules (FIG. 2).

The potency of the full-length form of BoNT/A, B and G wild types wasdetermined in the HDA by a dosage-effect-graph (FIGS. 3 and 6). Thepotency of the different full-length forms of BoNT/A, B and G singlemutants was subsequently determined in the HDA (FIG. 6) and plottedagainst the potency of the BoNT/B and G wild types by means of anapplied potency function (FIGS. 4 and 5). For example, the substitutionof the amino acids valine 1118 by aspartate or lysine 1192 by glutamatein BoNT/B results in a drastic reduction of the potency to <2%. Incontrast thereto, the mutation of the tyrosine 1183 in leucine orarginine, respectively, brings about a significant increase of thepotency of BoNT/B (FIG. 4). Modifying tyrosine 1256 to phenylalanine inBoNT/G results likewise in an increase in potency while the mutation ofglutamine 1200 in glutamate, lysine or tyrosine causes a considerabledecrease of the potency of BoNT/G (FIG. 5). In the case of BoNT/A,modifying serine 1207 to arginine or tyrosine brings about an increasein potency while the mutation of lysine 1260 to glutamate causes adrastic potency reduction of the BoNT/A (FIG. 6).

According to a preferred embodiment the transport protein exhibits anaffinity which is at least 15% higher or at least 15% lower than thenative neurotoxin. Preferably, the transport protein exhibits anaffinity which is at least 50% higher or lower, particularly preferredat least 80% higher or lower, and, in particular, at least 90% higher orlower than the native neurotoxin.

According to a preferred embodiment the modification of the HC takesplace in the region of the H_(C)-fragment of the given neurotoxin. Ifthe modification includes a substitution, deletion, insertion oraddition, the latter may be performed, for example, by specificmutagenesis, methods in this context being known to the person skilledin the art. The amino acids present in the native neurotoxin are in thiscontext modified either by naturally occurring or by not naturallyoccurring amino acids. Amino acids are, in principle, divided intodifferent physicochemical groups. Aspartate and glutamate belong to thenegatively-charged amino acids. Histidine, arginine and lysine belong tothe positively-charged amino acids. Asparagine, glutamine, serine,threonine, cysteine and tyrosine belong to the polar amino acids.Glycine, alanine, valine, leucine, isoleucine, methionine, proline,phenylalanine and tryptophane belong to the non-polar amino acids.Aromatic side groups are to be found among the amino acids histidine,phenylalanine, tyrosine and tryptophane. In general, it is preferred tosubstitute an amino acid by a different amino acid pertaining to anotherphysicochemical group.

According to a preferred embodiment of the invention, the transportprotein is a Botulinus neurotoxin serotype A to G. The amino acidsequences of the native neurotoxins can in this context be obtained frompublicly accessible databases as follows:

TABLE 1 Database numbers of the amino acid sequences and distribution ofthe sub-domains of the seven Botulinus neurotoxins. Database Number no.of SEQ of the HC the protein ID amino H_(C) BoNT sequence NO.: acidsH_(N) H_(CN) H_(CC) BoNT/ AAA23262 2 1296 449-866 867-1091 1092-1296 AAAM75961 2 AAQ06331 2 BTCLAB 2 P10845 3 1296 449-866 867-1091 1092-1296CAA36289 4 1296 449-866 867-1091 1092-1296 CAA51824 5 1296 449-866867-1091 1092-1296 I40645 5 Q45894 5 BoNT/ AAL11499 6 1291 442-855866-1078 1079-1291 B AAL11498 6 CAA73968 7 1291 442-855 866-10781079-1291 AAK97132 8 1291 442-855 866-1078 1079-1291 A48940 9 1291442-855 866-1078 1079-1291 AAA23211 9 P10844 9 BAC22064 10 1291 442-855866-1078 1079-1291 CAA50482 11 1291 442-855 866-1078 1079-1291 I40631 11BoNT/ A49777 12 1291 450-863 864-1092 1093-1291 C1 BAA14235 12 BAB7174913 CAA51313 12 S46431 12 P18640 14 1291 450-863 864-1092 1093-1291BAA08418 15 1280 450-863 864-1083 1084-1280 BAA89713 16 1280 450-863864-1083 1084-1280 BoNT/ CAA38175 17 1276 446-859 860-1079 1080-1276 DP19321 17 S11455 17 AAB24244 18 1276 446-859 860-1079 1080-1276 BAA0747719 1285 446-859 860-1088 1089-1285 S70582 19 BAA90661 20 1285 446-859860-1088 1089-1285 BoNT/ BAB86845 21 1252 423-842 843-1066 1067-1252 ECAA44558 21 S21178 21 CAA43999 22 1251 423-842 843-1066 1067-1251 Q0049622 CAA43998 23 1251 423-842 843-1066 1067-1251 JH0256 23 P30995 23 BoNT/1904210A 24 1274 440-860 861-1086 1087-1274 F AAA23263 24 I40813 24P30996 24 CAA73972 25 1280 440-861 862-1087 1088-1280 AAA23210 26 1278440-861 862-1084 1085-1278 CAA57358 26 CAA48329 27 1268 432-853 854-10751076-1268 S33411 27 BoNT/ CAA52275 28 1297 447-860 861-1086 1087-1297 GQ60393 28 S39791 29

With regard to the H_(c)-fragment of these Botulinus neurotoxins, theamino acids in the amino acid positions from

867 to 1296 of the C. botulinum neurotoxin serotype A,

866 to 1291 of the C. botulinum neurotoxin serotype B,

864 to 1291 or, respectively, 1280 of the C. botulinum neurotoxinserotype C1,

860 to 1276 or, respectively, 1285 of the C. botulinum neurotoxinserotype D,

843 to 1251 or, respectively, 1252 of the C. botulinum or C. butyricumneurotoxin serotype E,

861 to 1274, 862 to 1280 or, respectively, 1278 and 854 to 1268 of theC. botulinum or, respectively, C. baratii neurotoxin serotype F

861 to 1297 of the C. botulinum neurotoxin serotype G are preferred formodification.

It is, therefore, preferred to modify post-translationally, and/or add,and/or delete, and/or insert, and/or substitute by an either naturallyoccurring or not naturally occurring amino acid at least one amino acidin the aforesaid positions.

According to a preferred embodiment, the neurotoxin is Botulinusneurotoxin serotype A. In this case, preferably at least one amino acidin the positions threonine 1195, asparagine 1196, glutamine 1199, lysine1204, isoleucine 1205, leucine 1206, serine 1207, leucine 1209,aspartate 1213, leucine 1217, phenylalanine 1255, asparagine 1256,isoleucine 1258 and/or lysine 1260 of the Botulinus neurotoxin serotypeA protein sequences is modified post-translationally, and/or added,and/or deleted, and/or inserted and/or substituted by an eithernaturally occurring or not naturally occurring amino acid. The positionsasparagine 1196, glutamine 1199, serine 1207, phenylalanine 1255,isoleucine 1258 and/or lysine 1260 of the Botulinus neurotoxin serotypeA protein sequences are particularly preferred. In particular, thepositions serine 1207, substituted by arginine or tyrosine, and lysine1260, substituted by glutamate, are preferred.

According to a preferred embodiment, the neurotoxin is Botulinusneurotoxin serotype B. In this case, preferably at least one amino acidin the positions lysine 1113, aspartate 1114, serine 1116, proline 1117,valine 1118, threonine 1182, tyrosine 1183, phenylalanine 1186, lysine1188, glutamate 1191, lysine 1192, leucine 1193, phenylalanine 1194,phenylalanine 1204, phenylalanine 1243, glutamate 1245, lysine 1254,aspartate 1255 and tyrosine 1256 of the Botulinus neurotoxin serotype Bprotein sequences is modified post-translationally, and/or added, and/ordeleted, and/or inserted and/or substituted by an either naturallyoccurring or not naturally occurring amino acid. The positions valine1118, tyrosine 1183, glutamate 1191, lysine 1192, glutamate 1245 andtyrosine 1256 of the Botulinus neurotoxin serotype B protein sequencesare particularly preferred. In particular, the positions of tyrosine1183 and glutamate 1191, substituted by leucine, are preferred.

According to a further preferred embodiment, the neurotoxin is Botulinusneurotoxin serotype G. In this case, preferably at least one amino acidin the positions phenylalanine 1121, lysine 1123, alanine 1124, serine1125, methionine 1126, valine 1190, leucine 1191, serine 1194, glutamate1196, threonine 1199, glutamine 1200, leucine 1201, phenylalanine 1202,phenylalanine 1212, phenylalanine 1248, lysine 1250, aspartate 1251 andtyrosine 1262 of the Botulinus neurotoxin serotype G protein sequencesis modified post-translationally, and/or added, and/or deleted, and/orinserted and/or substituted by an either naturally occurring or notnaturally occurring amino acid. The positions methionine 1126, leucine1191, threonine 1199, glutamine 1200, lysine 1250 and tyrosine 1262 ofthe Botulinus neurotoxin serotype G protein sequences are particularlypreferred. In particular, the position tyrosine 1262, substituted byphenylalanine, is preferred.

The transport protein provided in the present invention exhibits anincreased or reduced specific affinity of its protein-binding domain, inparticular to molecules pertaining to the family of the synaptotagminsor the synaptic vesicle glycoproteins 2.

A further embodiment of the present invention relates to a compositioncontaining a transport protein according to the invention and at leastone intervening molecule (X). The intervening molecule may be a smallorganic molecule, a peptide or a protein; preferably covalently bondedto the transport protein by a peptide bond, an ester bond, an etherbond, a sulphide bond, a disulphide bond or a carbon-carbon-bond.

In addition, the intervening molecule includes all known therapeuticallyactive substances. Cytostatics, antibiotics, virustatics, but alsoimmunoglobulins are preferred in this context.

In a preferred embodiment, the protein is a protease, splitting one or aplurality of proteins of the release apparatus of neurotransmitters, theprotease being selected from the group of neurotoxins consisting of theLCs of the C. botulinum neurotoxins, in particular of the serotype A, B,C1, D, E, F and G or a proteolytically active fragment of the LC of a C.botulinum neurotoxin, in particular a neurotoxin of the serotype A, B,C1, D, E, F and G, the fragment exhibiting at least 0.01%, preferably atleast 5%, particularly preferably at least 50%, in particular at least90% of the proteolytic activity of the native protease. Preferably, thetransport protein and the protease are derived from the same C.botulinum neurotoxin serotype, in particular and preferably theH_(N)-domain of the transport protein and the protease are derived fromthe C. botulinum neurotoxin serotype A. The sequences of the proteasesare generally accessible at databases and the database numbers areapparent from Table 1. The proteolytic activity of the proteases isdetermined by way of substrate separation kinetics (see Binz et al.(2002), Biochemistry 41(6), 1717-23).

According to a further embodiment of the present invention, a processfor producing the transport protein is provided. In this case, in afirst step a nucleic acid coding for the transport protein is provided.The coding nucleic acid may represent in this context RNA, DNA ormixtures thereof. The nucleic acid may furthermore be modified withregard to its nuclease resistance, such as e.g. by insertingphosphorthioate bonds. The nucleic acid may be produced from a startingnucleic acid, the latter being accessible e.g. by cloning from genomicor cDNA-databases. Moreover, the nucleic acid may be produced directlyby solid phase synthesis. Suitable methods are known to the personskilled in the art. If one starts with a starting nucleic acid, aspecific modification, e.g. by locality-specific mutagenesis, may bebrought about, resulting in at least one addition, insertion, deletionand/or substitution on the amino acid level. The nucleic acid is thenlinked operatively to a suitable promoter. Suitable promoters forexpression in known expression systems are known to the person skilledin the art. The choice of promoter depends in this case on theexpression systems used for expression. In general, constitutivepromoters are preferred, but inducible promoters may likewise be used.The construct produced in this manner includes at least one part of avector, in particular regulatory elements, the vector being selected,for example, from X-derivates, adenoviruses, baculoviruses, vacciniaviruses, SV40-viruses and retroviruses. The vector is preferably capableof expressing the nucleic acid in a given host cell.

The invention further provides host cells, which contain the vector andare suitable for expressing the vector. Numerous prokaryotic andeukaryotic expression systems are known in the state of the art, thehost cells being selected, for example, from prokaryotic cells such asE. coli or B. subtilis, from eukaryotic cells such as S. cerevisiae andP. pastoris. Although higher eukaryotic cells, such as insect cells ormammal cells, may be used as well, host cells are neverthelesspreferred, which, like C. botulinum, do not possess a glycosylationapparatus.

According to a preferred embodiment the nucleic acid codes for theH_(C)-fragment of the C. botulinum neurotoxin. This nucleic acidcontains endonuclease-interfaces, flanking the nucleic acid coding forthe H_(C)-fragment, the endonuclease sites being compatible with thoseof other H_(C)-fragments of C. botulinum neurotoxins, in order to permittheir easy modular substitution in the gene coding for the transportprotein, while the similarity of the amino acid sequence is preserved.

If a composition according to the invention is provided, which, apartfrom the transport system, further contains at least one interveningmolecule, and this intervening molecule is a peptide or protein,functionalised either with a carboxyl-terminal cysteine or amercapto-group, then, in an analogous manner, as described before, thepeptide and/or protein may be produced recombinantly, for example byusing binary vectors or various host cells. If the same host cell isused for the expression both of the transport protein and the peptide orprotein, an intermolecular disulphide bond is preferably formed in situ.For a more efficient production in the same host cell, the nucleic acidcoding for the peptide or protein may also be translated with that ofthe transport protein in the same reading frame, so that a single-chainpolypeptide is produced. In this case, preferably an intramoleculardisulphide bond is formed in situ. For simple hydrolysis of the likewisepresent peptide cross-linking between the transport protein and thepeptide and/or protein, an amino acid sequence is inserted at theamino-terminus of the transport protein, which is either specificallyrecognised and split by the protease thrombin or by a specificendoprotease of the host cell.

Surprisingly, it was found that the insert-sequence CXXXZKTKSLVPRGSKBXXC(SEQ ID NO:1), with X signifying any desired amino acid and Z and Bbeing selected independently of each other from alanine, valine, serine,threonine and glycine, is split efficiently in vivo by an endogenousprotease of a bacterial host, preferably E. coli. The insertion of theinsert-sequence between the amino acid sequence of the transport proteinand a further peptide or protein therefore offers the advantage thatpost-treatment at a later stage, e.g. by thrombin, is not necessary. TheE. coli-strain E. coli K12 is particularly preferred.

Preferably, the insert-sequence forms part of a loop with 18 20,preferably amino acids.

If this is not possible, an appropriate intermolecular disulphide-bond,after separate purification of the transport protein and the protein,may subsequently be brought about by oxidation processes known to theperson skilled in the art. The peptide or protein may also be obtaineddirectly by synthesis or fragment condensation. Appropriate methods areknown to the person skilled in the art.

The transport protein and the peptide, or protein, respectively, aresubsequently purified. For this purpose methods are used, known to theperson skilled in the art, such as e.g. chromatography-methods orelectrophoresis.

A further embodiment of the present invention relates to thepharmaceutical composition, which includes the transport protein or acomposition and optionally a pharmaceutically acceptable excipient, adiluent and/or an additive.

The pharmaceutical composition is suitable for oral, intravenous,subcutaneous, intramuscular and topical administration. Intramuscularadministration is preferred in this context. A dosing unit of thepharmaceutical composition contains approximately 0.1 pg to 1 mg oftransport protein and/or the composition according to the invention.

The pharmaceutical composition is suitable to treat disorders ofneurotransmitter release and disorders such as hemi-facial spasms,spasmodic torticollis, blepharospasm, spasticities, dystonias, migraine,pain, disorders of the neck and lumbar vertebral column, strabism,hypersalivation, wound healing, snoring and depression.

A further embodiment of the present invention includes a cosmeticcomposition, containing a transport protein and a cosmeticallyacceptable excipient, diluent and/or additive. The cosmetic compositionis suitable for treating hyperhidrosis and facial wrinkles.

FIG. 1: Study of the in vitro bond of the wild type and mutated BoNT/BH_(C)-fragments to shortened GST-syt I and GST-syt II fusion proteins inthe presence or absence of complex gangliosides by means ofGST-pull-down assay.

FIG. 2: Study of the in vitro bond of the wild type and mutated BoNT/GH_(C)-fragments to shortened GST-syt I and GST-syt II fusion proteins inthe presence or absence of complex gangliosides by means ofGST-pull-down assay.

FIG. 3: Dosage-effect-graph of the BoNT/B and G wild types in the HDA.The applied potency functions permit a relative comparison of theparalysis times of single mutants with those of the associated wildtypes.

FIG. 4: Increase and decrease of the neurotoxicity of the BoNT/B singlemutants compared to the wild type in the HDA.

FIG. 5: Increase and decrease of the neurotoxicity of the BoNT/G singlemutants compared to the wild type in the HDA.

FIG. 6: Dosage-effect-graphs of the BoNT/A wild type and the BoNT/Asingle mutants in the HDA.

In detail, the present invention relates to a transport protein (Trapo),formed by modifying the HC of the neurotoxin produced by C. botulinum,preferably specifically binding to neurons, and preferably absorbedintracellularly by receptor-mediated endocytosis and translocated fromthe acid endosomal compartment into the cytosol of neurons. This proteinis used as a transporting means in order to introduce into the cellsproteases and other substances bound to the said transporting means,unable to penetrate physiologically into the plasma membrane and toreach the cytosol of nerve cells. The substrates of the proteases areintracellularly localised proteins and peptides participating in thetransmitter release. After separation of the substrates, the specificfunctions of the neurons are blocked; the cells themselves are notdamaged. One of these functions is exocytosis, bringing about theneurotransmitter release. If the release of transmitters is inhibited,the transmission of signals from cell to cell is blocked. For example,striated muscles are paralysed if the release of acetyl choline isinhibited at the neuromuscular contact point. This effect may be usedtherapeutically, if the transport protein is applied to nerve ends ofspastic or dystonic muscles. Other active substances are, for example,substances exhibiting anti-viral action. Conjugated with the transportprotein, they are of use for treating viral infections of the nervoussystem. The present invention also relates to the use of a transportprotein for inhibiting the release of neurotransmitters.

Transport proteins with a relatively low affinity bind to the nervecells, but are not absorbed by them. These transport proteins aretherefore suitable to serve as specific transporting means towards thesurface of the nerve cells.

If patients are treated with the progenitor toxins A and B from C.botulinum, the injection of these non-human proteins, despite the lowdosage, causes the formation of antibodies, so that the therapy shows noeffect and must therefore be stopped in order to prevent anaphylacticshock. By applying a substance with the same active mechanism having ahigher transport efficiency of the enzymatic activity, the dosage may belowered drastically and the formation of antibodies will not occur.These properties are attributed to the transport protein describedherein.

Although examples for application are given, the suitable mode ofapplication and the dosage is, in general, individually determined bythe treating physician. Such decisions are routinely made by eachphysician well versed in the relevant special field. Thus, the mode ofapplication and the dosage of the neurotoxin may e.g. be selected inaccordance with the invention described herein, based on criteria suchas the solubility of the selected neurotoxin or the intensity of thepain to be treated.

The treatment interval for the native progenitor toxins A and B from C.botulinum is currently three to four months on average. Prolonging thisinterval would reduce the risk of the formation of antibodies and allowa longer treatment period with BoNT. The increase of LC in the cytosolwould retard its decomposition and would thus also prolong the durationof efficacy. The transport protein described here exhibits a higheraffinity and absorption rate than the native HC.

The following example merely serves for elucidation and should not beunderstood in a limiting manner.

Material and Methods

Plasmid Construction and Preparation of Recombinant Proteins

Plasmids for E. coli expression of recombinant H_(C)-fragments of BoNT/Band BoNT/G as well as of the full-length form of BoNT/A, B and G withcarboxyl-terminal StrepTag for affinity purification were brought aboutby PCR-methods with suitable primers, chromosomal DNA coding for BoNT/A(AAA23262) BoNT/B (AAA23211) and BoNT/G (CAA52275) and the expressionvector pQe3 (Quiagen AG) serving as the starting vector. Shortenedvariations of rat-synaptotagmin I (syt I) (amino acids 1-53; amino acids1-82) and rat-synaptotagmin II (syt II) (amino acids 1-61; amino acids1-90) were cloned into the GST-coding vector pGEX-2T (AmershamBiosciences AB). The nucleic acid sequences of all plasmids wereconfirmed by DNA-sequencing. The recombinant H_(C)-fragments and thoseof the full-length form of BoNT were prepared at room temperature in theE. coli-strain M15 [pRep4] (Qiagen) during induction for ten hours andpurified on a StrepTactin-matrix (IBA GmbH) in accordance with themanufacturers instructions. The GST-fusion proteins obtained from E.coli BL21 were isolated with the aid of glutathione immobilised onsepharose micro-beads. Fractions containing the desired proteins werecombined and dialysed against Tris-NaCl-triton-buffer (20 mM Tris-HCl,150 mM NaCl, 0.5% Triton X-100, pH 7.2).

GST-Pull-Down Assay

GST-fusion proteins (0.12 nmol each), which had been immobilised on 10μl GT-sepharose micro-beads, were incubated at 4° C. for 2 h withH_(C)-fragments (0.1 nmol) in the absence or in the presence of a bovinebrain-ganglioside-mixture (18% GM1, 55% GD1a, 10% GT1b, 2% othergangliosides; Calbiochem; 20 μg each) in a total volume of 180 μlTris-NaCl-triton-buffer. The micro-beads were collected by centrifuging,the supematant was removed and the separated micro-beads were in eachcase rinsed three times with 400 μl of the same buffer. The rinsedpellet fractions were boiled in SDS-sample buffer and studied, togetherwith the supernatant fractions, by SDS-PAGE and Coomassie blue staining.

The BoNTB wild type binds only in the presence of complex gangliosidesand synaptotagmin I with transmembrane domain, while synaptotagmin IIbinds with or without transmembrane domain as well as in the presence orin the absence of complex gangliosides. By specifically substitutingamino acids within the protein receptor binding site of BoNT/B it waspossible to significantly increase (E1191L; Y1183L) or decrease (V1118D;K1192E) the interaction between both synaptotagmin molecules (FIG. 1).

For the BoNT/G wild type it was shown that binding to synaptotagmin Iand synaptotagmin II, in each case with or without transmembrane domain,is taking place both in the presence as well as in the absence ofcomplex gangliosides. By specifically substituting amino acidshomologous to BoNT/B, within the protein receptor binding site ofBoNT/G, it was possible to significantly increase (Y1262F) or decrease(Q1200E) the interaction between both synaptotagmin molecules (FIG. 2).

By detecting the bond of the recombinant H_(C)-fragments of BoNT/B and Gto isolated, immobilised gangliosides, it was possible to exclude damageto the function of the neighbouring ganglioside-binding pocket by themutations introduced into the syt-binding pocket and to draw adequateconclusions to an intact tertiary structure of the H_(C)-fragment. Theseresults were supported by CD-spectroscopic studies as well as by thermaldenaturation experiments, likewise displaying intact tertiary structuresof the mutated H_(C)-fragments of BoNT/B and G.

Mouse Hemidiaphraam Assay (HDA)

The neurotoxicity of the BoNT/A, B and G-mutants was determined asdescribed by Habermann et al., Naunyn Schmiedeberg's Arch. Pharmacol.311 (1980), 33-40.

The potency of the full-length form of BoNT/A, B and G wild types wasdetermined in the HDA by a dosage-effect-graph (FIGS. 3 and 6). Thepotency of the different full-length forms of BoNT/A, B and G singlemutants was subsequently determined in the HDA (FIG. 6) and plottedagainst the potency of the BoNT/B and G wild types by means of anapplied potency function (FIGS. 4 and 5). Thus, the substitution of theamino acids valine 1118 by aspartate or lysine 1192 by glutamate inBoNT/B results in a drastic reduction of the potency to <2%. In contrastthereto, the mutation of the tyrosine 1183 in leucine or arginine,respectively, brings about a significant increase of the potency ofBoNT/B (FIG. 4). Modifying tyrosine 1256 to phenylalanine in BoNT/Gresults likewise in an increase in potency while the mutation ofglutamine 1200 in glutamate, lysine or tyrosine causes a considerabledecrease of the potency of BoNT/G (FIG. 5). In the case of BoNT/A,modifying serine 1207 to arginine or tyrosine brings about an increasein potency while the mutation of lysine 1260 to glutamate causes adrastic potency reduction of the BoNT/A (FIG. 6).

The invention claimed is:
 1. A transport protein comprising a modifiedClostridium botulinum serotype A neurotoxin heavy chain, the heavy chaincomprising: (A) a H_(N)-fragment of Clostridium botulinum serotype A ofSEQ ID NOs.: 2, 4 or 5; and (B) a H_(C)-fragment of Clostridiumbotulinum serotype A of SEQ ID NOs.: 2, 4 or 5, modified by thesubstitution of an amino acid at positions 1195, 1196, 1199, 1204, 1205,1206, 1207, 1213, 1217, 1258, and/or 1260 with either a naturallyoccurring or a non-naturally occurring amino acid, wherein theH_(C)-fragment comprises a H_(CC)-fragment and a H_(CN)-fragment, andwherein the H_(CC)-fragment binds to a nerve cell with a higher or loweraffinity than native Clostridium botulinum serotype A neurotoxin.
 2. Theprotein of claim 1, which binds to a nerve cell with an affinity that isat least 15% higher or lower than its binding affinity for nativeClostridium botulinum serotype A neurotoxin.
 3. The protein of claim 1,which binds to synaptic vesicle glycoprotein 2 (SV2).
 4. The protein ofclaim 3, which binds to SV2A, SV2B or SV2C.
 5. The protein of claim 1,wherein the H_(C)-fragment is modified by the substitution of an aminoacid at positions 1196, 1199, 1207, 1258, and/or 1260 of SEQ ID NOs.: 2,4 or 5; and wherein the substitution is with either a naturallyoccurring or a non-naturally occurring amino acid.
 6. The protein ofclaim 5, wherein the amino acid at position 1207 is substituted witharginine or tyrosine.
 7. The protein of claim 5, wherein the amino acidat position 1260 is substituted with glutamate.
 8. The protein of claim1, which protein is covalently linked to a clostridial translocationdomain.
 9. The protein of claim 8, which protein is covalently linked toa clostridial neurotoxin protease.
 10. The protein of claim 9, whichprotein includes an amino acid sequence that is recognized by, and maybe cleaved by, a protease to provide a di-chain polypeptide, wherein theclostridial translocation domain and the clostridial neurotoxin proteaseare linked together by a disulfide-bridge.
 11. The protein of claim 10,wherein the amino acid sequence is a native clostridial amino acidsequence or a non-clostridial amino acid sequence.
 12. A method fortreating an individual suffering from a disease or disorder selectedfrom hemi-facial spasm, spasmodic torticollis, blepharospasm,spasticities, dystonias, migraine, pain, disorders of the neck andlumber vertebral column, strabism, hypersalivation, snoring, woundhealing, and depressive disorders, the method comprising administering atherapeutically effective amount of a protein according to claim 1 tothe individual.
 13. A method for treating an individual suffering from acosmetic indication selected from hyperhidrosis and facial wrinkles, themethod comprising administering a therapeutically effective amount of atransport protein according to claim 1 to the individual.
 14. A processfor producing the protein of claim 1, which protein is produced byrecombinant expression.